- Email: [email protected]
reperfusion injury
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Oral administration of cilostazol improves survival rate after rat liver ischemia/reperfusion injury Taku Fujii, MD,a Hideaki Obara, MD, PhD,b,* Kentaro Matsubara, MD, PhD,b Naoki Fujimura, MD, PhD,b Hiroshi Yagi, MD, PhD,b Taizo Hibi, MD, PhD,b Yuta Abe, MD, PhD,b Minoru Kitago, MD, PhD,b Masahiro Shinoda, MD, PhD,b Osamu Itano, MD, PhD,b Minoru Tanabe, MD, PhD,c Yohei Masugi, MD, PhD,d Michiie Sakamoto, MD, PhD,d and Yuko Kitagawa, MD, PhDb a
Department of Surgery, Hiratsuka City Hospital, Kanagawa, Japan Department of Surgery, Keio University School of Medicine, Tokyo, Japan c Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan d Department of Pathology, Keio University School of Medicine, Tokyo, Japan b
article info
abstract
Article history:
Background: Cilostazol is a type III phosphodiesterase inhibitor used to treat the symptoms
Received 25 October 2016
of intermittent claudication. Recent studies have shown that cilostazol decreases
Received in revised form
ischemia/reperfusion (I/R) injury in several organs.
7 February 2017
Materials and methods: We evaluated the effects of cilostazol in a rat model of liver I/R injury.
Accepted 16 February 2017
Thirty male Wistar rats with liver I/R injury were divided into a cilostazol or saline (control)
Available online 23 February 2017
group (n ¼ 15 each). Each rat was orally administered cilostazol or saline for 3 d before I/R injury. Liver I/R injury was induced via 1 h of warm ischemia of the median and left lateral
Keywords:
liver lobes, followed by 3 h of reperfusion. The rats were then euthanized. Serum aspartate
Cilostazol
aminotransferase, alanine aminotransferase, interleukin (IL)-1b, IL-6, and tumor necrosis
Alanine aminotransferase
factor-a levels were measured. The ManneWhitney U test was used to compare the dif-
Aspartate aminotransferase
ferences between the treatment groups. Histologic examination was performed on the liver
Hepatocyte injury
tissues. We also conducted a survival study to confirm the effect of cilostazol on the
Partial ischemia/reperfusion injury
mortality rate in rats. For the survival study, a liver I/R injury model with an ischemia time
Phosphodiesterase III inhibitor
of 1.5 h was used, and the rats were observed for 1 wk. Results: Serum aspartate aminotransferase, alanine aminotransferase, IL-1b, and IL-6 levels were significantly lower in the cilostazol group than in the saline group. Treatment with cilostazol significantly improved pathological findings associated with liver I/R injury and increased survival rate compared to that in controls. Conclusions: Cilostazol reduced mortality and alleviated the effects of liver I/R injury in Wistar rats. ª 2017 Elsevier Inc. All rights reserved.
Funding: This study was supported in part by Grant-in-Aid for Scientific Research (grant number 15k10172) of the Japan Society for the Promotion of Science (Tokyo, Japan). * Corresponding author. Department of Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Tel.: þ81 3 3353 1211; fax: þ81 3 3355 4707. E-mail address: [email protected] (H. Obara). 0022-4804/$ e see front matter ª 2017 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2017.02.020
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Introduction
Surgical procedure
Ischemia/reperfusion (I/R) injury is a serious complication of liver resection and liver transplantation and a major contributor to the associated morbidities.1 Alleviating liver I/R injury can improve survival rate by minimizing postoperative liver injury. Data from studies in laboratory animals have suggested that I/R injury can be alleviated by ischemic preconditioning and postconditioning, some pharmacologic agents, gene therapy, and machine perfusion; however, none of these interventions have been recommended for use in clinical practice.2 I/R injury is typified by an inflammatory response; liver I/R injury involves a complex web of interactions between Kupffer cells, CD4þ lymphocytes, neutrophils, and hepatocytes, as well as various cytokines, chemokines, and complement proteins.3 Over the last 20 y, cilostazol (6-[4-(1-cyclohexyl-1h-tetrazol-5-yl) butyloxy]-3, 4-dihydroquinolin-2(1H)-one) has been used worldwide to treat the symptoms of intermittent claudication caused by peripheral arterial disease. The InterSociety Consensus for the Management of Peripheral Arterial Disease (TASC II) has suggested cilostazol as the first-line drug for the relief of claudication symptoms because it improves treadmill exercise performance and quality of life.4 Cilostazol is a phosphodiesterase III inhibitor that suppresses degradation of cyclic adenosine monophosphate (cAMP), resulting in an increased concentration of cAMP in platelets and blood vessels, which leads to inhibition of platelet aggregation and vasodilation, respectively.5,6 In addition, cilostazol inhibits leukocyte adhesion to the endothelium7 and appears to tighten the endothelial barrier in situ by partly inhibiting cAMP-degrading enzymes in the endothelium.8 A study reported that cilostazol suppresses neointimal formation by suppressing proliferation of vascular smooth muscle cells.9 Recent studies have shown that cilostazol alleviates I/R injury in the brain, spinal cord, and small bowel.10e12 Previously, we investigated the mechanism of liver I/R injury.13e15 Based on our findings, in the present study, we hypothesized that cilostazol is beneficial for the treatment of liver I/R injury. We also examined the effect of cilostazol on the survival rate in rats with liver I/R injury.
Liver I/R injury was induced in the rats according to a previously reported procedure.16 Briefly, a midline incision was made after shaving the abdomen, and the abdominal cavity was then exposed with the aid of retractors. Next, the left hepatoduodenal ligament containing the hepatic artery, portal vein, and bile duct of the left lateral and median liver lobes was clamped for 60 or 90 min with a microvascular clamp to induce partial (70%) warm ischemia. The 90-min ischemia model was used to determine survival and the 60-min ischemia model was for all other experiments; 60 min of partial ischemia is nonlethal.17 Thereafter, the clip was removed to initiate hepatic reperfusion, and the abdominal cavity was closed. All the procedures were performed under general anesthesia induced with isoflurane. The rats were subjected to warm ischemia for 60 min followed by 3 h of reperfusion. The animals were then euthanized by total blood collection from the aorta by exsanguination. Liver tissues and blood samples were taken for analysis.
Materials and methods Animals Male Wistar rats aged 8 wk (weighing 250-300 g) were purchased from Japan SLC, Inc (Hamamatsu, Shizuoka, Japan) and housed in a temperature- and humiditycontrolled room under a 12-h light/dark cycle. The animals were given free access to water and food. All experiments were performed in accordance with the guidelines for the use of experimental animals by the National Institutes of Health and were approved by the Laboratory Animal Care and Use Committee of Keio University School of Medicine.
Experimental protocol The rats were randomly assigned to either a control group (I/R þ saline, n ¼ 15) or cilostazol group (I/R þ cilostazol, n ¼ 15). Cilostazol (Otsuka Pharmaceutical, Tokushima, Japan) was dissolved in a 0.5% carboxymethylcellulose sodium salt (Wako Pure Chemical Industries Ltd, Osaka, Japan) solution at a concentration of 10 mg/mL. Each rat was administered cilostazol (20 mg/kg/d) or saline (0.5 mL/ body/d) through a 3-Fr feeding tube (NIPRO, Osaka, Japan) into the stomach. These administrations were started 3 d before the surgery. On the day of the surgery, cilostazol or saline was administered 30 min before the induction of ischemia.18 Cilostazol or saline was administered 4 times before the surgery was performed (Fig. 1).
Laboratory measurements Blood samples were obtained from the aorta at the end of the experimental protocol and centrifuged at 3000 rpm for 10 min to obtain serum, which was immediately stored at 80 C. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were determined using ultraviolet spectrophotometric and enzymatic assays. We also measured serum interleukin (IL)-1b, IL-6, and tumor necrosis factor-a (TNF-a) levels using an R&D Rat Magnetic Luminex Screening Assay Kit (R&D Systems Inc, Minneapolis, MN).
Histologic examination Liver tissues for histologic examination were sampled from the left and median liver lobes at the end of the experimental protocol. The specimens were fixed in a 10% buffered formaldehyde solution, embedded in paraffin, and stained with hematoxylin and eosin. The tissue samples were sectioned into 5-mm-thick pieces for analysis. Histologic examination was performed based on randomly selected tissue sections (n ¼ 5 from each group) using the scoring system proposed by
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fujii et al effect of cilostazol in liver i/r injury
saline
saline
saline
I/R injury
saline
0
1
2
Analysis of survival
3
Time after I/R injury (days) 0 CZ
CZ
0
1
2
2
6
7
6
7
CZ I/R injury
CZ
1
Analysis of survival
3
Time after I/R injury (days) 0
1
2
Fig. 1 e Experimental protocol. Rats were administered cilostazol (20 mg/kg/d) or saline (0.5 mL/body/d) via a feeding tube into the stomach once per day. These administrations were started 3 d before surgery. On the day surgery was performed, cilostazol or saline was administered 30 min before the induction of ischemia. The survival rate after inducing hepatic ischemia/reperfusion (I/R) was assessed every 24 h until 7 d after surgery.
Suzuki et al.19; specifically, the degree of sinusoidal congestion (none, minimal, mild, moderate, or severe), liver cell vacuolization (none, minimal, mild, moderate, or severe), and necrosis (none, single cell necrosis, < 30%, 30%-60%, or > 60%) in the tissues was determined. The histologic changes were scored from 0 to 4, and the total score, ranging from 0 to 12, was determined for each sample. All the sample slides were evaluated by the same pathologist, who was blinded to the corresponding control samples.
Survival analysis Eighteen Wistar rats (control, n ¼ 10; cilostazol, n ¼ 8) were used for a 7-d study on survival rate. Liver I/R injury was induced in the rats by subjecting them to warm ischemia for 90 min. After hepatic reperfusion, the rats recovered in individual cages and were fed a standard chow diet. Survival rate after the liver I/R injury was assessed every 24 h until 7 d after the operation (Fig. 1). The same cilostazol dosage (20 mg/kg/d) was administered in all the experiments.
Statistical analysis All results are expressed as means standard error of the mean. We performed between-group comparisons using either the ManneWhitney U test or Student t-test. A P value < 0.05 was considered statistically significant. We used the Kaplan-Meier analysis and log-rank testing to assess the
survival rate. All the statistical analyses were performed using SPSS 22 software (SPSS Inc, Chicago, IL).
Results Cilostazol preserves liver function To determine whether cilostazol alleviates liver I/R injury, cilostazol was administered to the rats before inducing warm I/R. As illustrated in Figure 2, serum AST (cilostazol versus control, 2427 1375 IU/L versus 4080 1870 IU/L; P ¼ 0.016) and ALT (cilostazol versus control, 2149 1299 versus 3513 1513 IU/L; P ¼ 0.023) levels at 3 h after hepatic reperfusion were significantly (P < 0.05) lower in the cilostazol group than in the control group. Therefore, treatment with cilostazol significantly alleviated liver I/R injury as indicated by the lowered levels of liver enzymes in the serum.
Cilostazol suppresses the expression of inflammatory cytokines in liver I/R injury Serum concentrations of IL-1b (cilostazol versus control, 31.7 26.2 pg/mL versus 49.0 19.0 pg/mL; P ¼ 0.013) and IL-6 (cilostazol versus control, 477 519 pg/mL versus 792 342 pg/ mL; P ¼ 0.020) were significantly lower in the cilostazol group than in the control group. However, no statistically significant differences were observed between serum TNF-a levels in the 2 groups (cilostazol versus control, 45.2 23.4 pg/mL versus 43.0 26.1 pg/mL; P ¼ 0.870; Fig. 3).
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Fig. 2 e Serum AST and ALT levels. Serum concentrations of AST and ALT were determined 3 h after hepatic reperfusion. The data are presented as means ± standard error of the mean (n [ 15 in each group).
Cilostazol improves the pathologic findings in liver I/R injury Histologic changes observed in the liver tissues are shown in Figure 4. In the control group, necrosis and sinusoidal congestions were evident from the periportal area to the midzonal area. In addition, vacuolization was apparent in the hepatocytes at the periphery of the necrotic area. In the cilostazol group, mild-to-moderate vacuolization and mild sinusoidal congestion were observed; however, no necrotic hepatocytes were observed (Fig. 4). The Suzuki scores for assessing liver damage were 3.4 1.1 and 6.0 2.1 for the cilostazol and saline control groups, respectively. The data indicate that liver damage was significantly (P < 0.05) alleviated by cilostazol. In particular, the Suzuki score for cytoplasmic vacuolization in the cilostazol group was significantly lower than that of the control group (cilostazol versus control, 1.4 0.5 versus 2.2 0.4; P ¼ 0.035). However, differences between Suzuki scores for sinusoidal congestion and necrosis in the 2 treatment groups were not statistically significant (Table).
Cilostazol increases the survival rate after liver I/R injury Treatment with cilostazol significantly improved the survival rate of rats after induction of liver I/R injury. The 7-d survival
rates were 63% and 100% in the saline control and cilostazol groups, respectively (P ¼ 0.038; Fig. 5).
Discussion Cilostazol is a quinolone compound that has antiplatelet effects. It acts directly on endothelial cells to inhibit the expression of adhesion molecules and inhibits neutrophil adhesion to the endothelium.20 After administration, cilostazol elevates the concentration of cAMP in endothelial cells and hepatocytes, which leads to the protection of endothelial cells and the maintenance of hepatic microcirculation and hepatocellular integrity.21e23 In rodent models, some studies have shown that I/R injury is alleviated through the cell protective effect of cilostazol in the kidney, heart, small intestine, limbs, muscles, spinal cord, cerebrum, and retina.12,24e28 It has been recently reported that intraperitoneally administered cilostazol alleviates hepatic I/R injury in mice.29 Although cilostazol was administered orally and intraperitoneally to rodents during these studies; clinically, it is only administered orally. In the present study, we analyzed the effects of oral cilostazol on survival rate and alleviation of hepatic injury in a rat model of partial warm hepatic I/R injury.
Fig. 3 e Serum levels of IL-1b, IL-6, and TNF-a. Serum concentrations of IL-1b, IL-6, and TNF-a were determined 3 h after hepatic reperfusion. The data are presented as means ± standard error of the mean (n [ 15 in each group).
fujii et al effect of cilostazol in liver i/r injury
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Fig. 4 e Histologic examination of the liver after I/R injury. Histologic analysis was performed on formalin-fixed liver sections (panels A and C are from the control group, whereas panels B and D are from the cilostazol group). The liver sections were obtained from the rats after they were subjected to partial hepatic ischemia for 60 min followed by 3 h of reperfusion. Representative images are shown. C, central vein and P, portal vein. Scale bars: 500 mm (A and B) and 100 mm (C and D).(Color version of figure is available online.)
Serum AST and ALT levels are the most objective indices of hepatocyte injury and are reported to markedly reflect the degree of hepatic I/R injury.30 Results of the present study show significant decreases in serum AST and ALT levels 3 h after reperfusion in the cilostazol group, suggesting that cilostazol alleviates hepatocyte injury. IL-1b and IL-6 are proinflammatory cytokines produced in Kupffer cells and hepatocytes after hepatic I/R injury.31 IL-1b is known to be closely associated with T cells/macrophages/neutrophils.32 In the present study, we observed significantly lower levels of serum IL-1b and IL-6 in the cilostazol group than in the saline group. These data suggest that alleviation of hepatic I/R injury
Table e Analysis of Suzuki scores. Histological changes
Control
Cilostazol
P value
Sinusoidal congestion
2.6 0.89
1.8 0.45
0.111
Cytoplasmic vacuolization
2.2 0.45
1.4 0.55
0.035
Necrosis
1.2 1.3
0.2 0.45
0.143
Total score
6.0 2.1
3.4 1.14
0.042
Estimation of the Suzuki score, which is representative of hepatocellular damage, was carried out 3 h after hepatic reperfusion. (n ¼ 5 in each group). Values are means standard error.
by cilostazol is mediated by the suppression of IL-1b and IL-6 expression levels. TNF-a expressed in Kupffer cells mediates hepatic I/R injury by inducing apoptosis and deterioration of the microcirculation through enhanced expression of cell adhesion molecules, which are induced by the generation of reactive oxygen species.31 However, in the present study, no significant differences were observed between serum TNF-a levels in the cilostazol and control groups. A study by Kim et al. reported that TNF-a levels peak after 6 h.33 Therefore, TNF-a levels at 6 h after the reperfusion in this model would be a next interesting. Hepatocellular vacuolization is a characteristic of cold hepatic I/R injury in rats. It has been reported that cilostazol suppresses the activation of endothelial cells and alleviates hepatocyte injury to reduce hepatocellular vacuolization.34 In contrast, in rat models of warm hepatic I/R injury, degeneration of nonparenchymal cells with vacuolization has been observed. Hepatic warm ischemia is poorly tolerated and rapidly leads to the death of sinusoidal endothelial cells and hepatocytes.23 The results of our study show that cilostazol improves these pathologic findings. Particularly, cytoplasmic vacuolization was markedly improved. This suggests that cilostazol alleviates hepatic I/R injury at the hepatocellular level. Other phosphodiesterase III inhibitors, including milrinone, olprinone, and amrinone are primarily used for managing cardiovascular diseases; however, they have been
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Fig. 5 e Analysis of survival rate after liver I/R injury. Survival rate was determined up to 7 d after 90 min of partial hepatic artery, portal vein, and bile duct clamping followed by reperfusion in rats (control group, n [ 8; cilostazol group, n [ 10).
shown to alleviate hepatic I/R injury in the liver and kidney.35e37Preconditioning is a technique conducted before hepatic ischemia to relieve I/R injury, whereas postconditioning is a protective procedure performed after I/R.38 Amrinone and milrinone have been noted to exert preconditioning and postconditioning effects in rats.39 Therefore, cilostazol treatment may result in a postconditioning effect in addition to the preconditioning effect observed in the present study. The data we obtained indicate that cilostazol improves survival rate after hepatic I/R injury. Other agents that have been observed to improve survival rate after hepatic I/R injury in rodent models include lithium and amrinone.37,40 Mortality may not reflect drug effects; rather, it may reflect events that are not ascertainable. Ischemia for 60 min is nonlethal17; therefore, we applied ischemia for 90 min to determine survival. In our preliminary experiments, survival rates were 100% 7 d after ischemia for 60 min. In the present study, the dosage of cilostazol administered to the rats was 20 mg/kg/d, which falls within the range of cilostazol dosages (10-40 mg/kg/d) administered to rats in previous studies.41,42 A previous study reported that oral administration of cilostazol at a dose of 10 mg/kg in rats is equivalent to a 100-mg/kg single dose in humans.43 Because the clinical dosage of cilostazol in humans is 200 mg/d, we administered 20 mg/kg/d to the rats. In the present study, the duration of induced hepatic partial ischemia was 60 min; however, previous studies have indicated that the extent of hepatic dysfunction is mild when the duration of ischemia is < 60 min.33,44 Pathologic findings were less remarkable for reperfusion lasting 3 h or less after ischemia; vacuolization, in
particular, remained relatively unchanged. However, when the reperfusion duration is > 3 h, necrosis and apoptosis become marked.44 We performed hepatic reperfusion over a period of 3 h in the present study and observed a significant improvement in vacuolization in the cilostazol group. In our preliminary experiments, we fixed the ischemic time to 1 h, and blood was collected 3, 6, 12, and 24 h after reperfusion. Hepatocyte damage, as shown by deviations in liver enzymes, peaked at 3 h. Consequently, we set the time after reperfusion at 3 h. According to Ritschel’s theory, drug concentrations reach a steady state when they are continuously administered 5 times longer than the half-life and the dose interval is less than 3 times the half-life. The half-life of cilostazol is 10-13 h; therefore, the time required for cilostazol to reach a steady state was estimated to be 50-65 h. We administered cilostazol for 3 d before reperfusion (72 h), and the experiment was conducted after the drug concentration reached a steady state. Although several agents have been reported to be effective in animal models of hepatic I/R injury, none have been clinically used in humans.31 Cilostazol is used clinically for the management of peripheral artery disease and for the prevention of cerebral infarction. Furthermore, the safety of cilostazol after oral administration has been established. Therefore, cilostazol may be a useful prophylactic against hepatic I/R injury. The primary limitation of the present study is the use of a rat model of partial (70%) hepatic ischemia induced by blocking blood flow to the median and left liver lobes to avoid enteric congestion. Therefore, the blood supply from the portal vein to nonischemic areas of the liver was higher than the blood supply to ischemic areas after reperfusion. This may have affected the extent of hepatic injury and rate of liver regeneration.45 In conclusion, the results of our study showed that oral administration of cilostazol before hepatic ischemia alleviates hepatic I/R injury in rats by protecting against hepatocyte injury and improving liver function, which can increase survival. Although further studies are required to understand the detailed mechanism by which cilostazol alleviates hepatic I/R injury, cilostazol may be a potential therapeutic agent for preventing hepatic I/R injury.
Acknowledgment The authors are grateful to Drs Yuko Matsuda and Kazumasa Fukuda for their invaluable technical assistance. Authors’ contributions: H.O. and T.F. were responsible for the conception and design of the project as well as collection of data. T.F., H.O., and M.S. analyzed the data. Y.M. and M.S. assessed the pathological findings. T.F., H.O., K.M., and N.F. were responsible for writing and revising the manuscript. T.F., H.O., K.M., N.F., H.Y., T.H., Y.A., M.K., M.S., O.I., M.T., Y.M., M.S., and Y.K. gave final approval of the manuscript.
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article.
fujii et al effect of cilostazol in liver i/r injury
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34. von Heesen M, Muller S, Keppler U, et al. Preconditioning by cilostazol protects against cold hepatic ischemia-reperfusion injury. Ann Transpl. 2015;20:160e168. 35. Yamaguchi K, Kawahara T, Kumakura S, et al. Effect of olprinone, a phosphodiesterase III inhibitor, on hepatic ischemia-reperfusion injury in rats. Shock. 2010;33:436e441. 36. Nishiki T, Kitada H, Okabe Y, et al. Effect of milrinone on ischemia-reperfusion injury in the rat kidney. Transpl Proc. 2011;43:1489e1494. 37. Kucuk C, Akcan A, Akyyldyz H, Akgun H, Muhtaroglu S, Sozuer E. Effects of amrinone in an experimental model of hepatic ischemia-reperfusion injury. J Surg Res. 2009;151:74e79. 38. de Rougemont O, Lehmann K, Clavien PA. Preconditioning, organ preservation, and postconditioning to prevent ischemia-reperfusion injury to the liver. Liver Transpl. 2009;15:1172e1182. 39. Toyoda T, Tosaka S, Tosaka R, et al. Milrinoneinduced postconditioning reduces hepatic ischemia-reperfusion injury in rats: the roles of phosphatidylinositol 3-kinase and nitric oxide. J Surg Res. 2014;186:446e451.
40. Liu A, Fang H, Dahmen U, Dirsch O. Chronic lithium treatment protects against liver ischemia/reperfusion injury in rats. Liver Transpl. 2013;19:762e772. 41. Nakamura T, Houchi H, Minami A, et al. Endotheliumdependent relaxation by cilostazol, a phosphodiesterase III inhibitor, on rat thoracic aorta. Life Sci. 2001;69:1709e1715. 42. Iida H, Iida M, Takenaka M, Fukuoka N, Dohi S. Comparative effects of cilostazol and aspirin on the impairment of endothelium-dependent cerebral vasodilation caused by acute cigarette smoking in rats. J Thromb Thrombolysis. 2010;29:483e488. 43. Akiyama H, Kudo S, Shimizu T. The absorption, distribution and excretion of a new antithrombotic and vasodilating agent, cilostazol, in rat, rabbit, dog and man. Arzneimittelforschung. 1985;35:1124e1132. 44. Batkai S, Osei-Hyiaman D, Pan H, et al. Cannabinoid-2 receptor mediates protection against hepatic ischemia/ reperfusion injury. FASEB J. 2007;21:1788e1800. 45. Furuchi K, Usami M, Ohyanagi H, Saitoh Y. Inhibitory effect of portal pooling, bacterial translocation, and Kupffer cell activation on hepatic regeneration after partial hepatectomy by repeated portal triad cross clamping in rats. Jpn J Gastroenterol. 1993;90:3006e3017.
Available online at www.sciencedirect.com
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Oral administration of cilostazol improves survival rate after rat liver ischemia/reperfusion injury Taku Fujii, MD,a Hideaki Obara, MD, PhD,b,* Kentaro Matsubara, MD, PhD,b Naoki Fujimura, MD, PhD,b Hiroshi Yagi, MD, PhD,b Taizo Hibi, MD, PhD,b Yuta Abe, MD, PhD,b Minoru Kitago, MD, PhD,b Masahiro Shinoda, MD, PhD,b Osamu Itano, MD, PhD,b Minoru Tanabe, MD, PhD,c Yohei Masugi, MD, PhD,d Michiie Sakamoto, MD, PhD,d and Yuko Kitagawa, MD, PhDb a
Department of Surgery, Hiratsuka City Hospital, Kanagawa, Japan Department of Surgery, Keio University School of Medicine, Tokyo, Japan c Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan d Department of Pathology, Keio University School of Medicine, Tokyo, Japan b
article info
abstract
Article history:
Background: Cilostazol is a type III phosphodiesterase inhibitor used to treat the symptoms
Received 25 October 2016
of intermittent claudication. Recent studies have shown that cilostazol decreases
Received in revised form
ischemia/reperfusion (I/R) injury in several organs.
7 February 2017
Materials and methods: We evaluated the effects of cilostazol in a rat model of liver I/R injury.
Accepted 16 February 2017
Thirty male Wistar rats with liver I/R injury were divided into a cilostazol or saline (control)
Available online 23 February 2017
group (n ¼ 15 each). Each rat was orally administered cilostazol or saline for 3 d before I/R injury. Liver I/R injury was induced via 1 h of warm ischemia of the median and left lateral
Keywords:
liver lobes, followed by 3 h of reperfusion. The rats were then euthanized. Serum aspartate
Cilostazol
aminotransferase, alanine aminotransferase, interleukin (IL)-1b, IL-6, and tumor necrosis
Alanine aminotransferase
factor-a levels were measured. The ManneWhitney U test was used to compare the dif-
Aspartate aminotransferase
ferences between the treatment groups. Histologic examination was performed on the liver
Hepatocyte injury
tissues. We also conducted a survival study to confirm the effect of cilostazol on the
Partial ischemia/reperfusion injury
mortality rate in rats. For the survival study, a liver I/R injury model with an ischemia time
Phosphodiesterase III inhibitor
of 1.5 h was used, and the rats were observed for 1 wk. Results: Serum aspartate aminotransferase, alanine aminotransferase, IL-1b, and IL-6 levels were significantly lower in the cilostazol group than in the saline group. Treatment with cilostazol significantly improved pathological findings associated with liver I/R injury and increased survival rate compared to that in controls. Conclusions: Cilostazol reduced mortality and alleviated the effects of liver I/R injury in Wistar rats. ª 2017 Elsevier Inc. All rights reserved.
Funding: This study was supported in part by Grant-in-Aid for Scientific Research (grant number 15k10172) of the Japan Society for the Promotion of Science (Tokyo, Japan). * Corresponding author. Department of Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Tel.: þ81 3 3353 1211; fax: þ81 3 3355 4707. E-mail address: [email protected] (H. Obara). 0022-4804/$ e see front matter ª 2017 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2017.02.020
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Introduction
Surgical procedure
Ischemia/reperfusion (I/R) injury is a serious complication of liver resection and liver transplantation and a major contributor to the associated morbidities.1 Alleviating liver I/R injury can improve survival rate by minimizing postoperative liver injury. Data from studies in laboratory animals have suggested that I/R injury can be alleviated by ischemic preconditioning and postconditioning, some pharmacologic agents, gene therapy, and machine perfusion; however, none of these interventions have been recommended for use in clinical practice.2 I/R injury is typified by an inflammatory response; liver I/R injury involves a complex web of interactions between Kupffer cells, CD4þ lymphocytes, neutrophils, and hepatocytes, as well as various cytokines, chemokines, and complement proteins.3 Over the last 20 y, cilostazol (6-[4-(1-cyclohexyl-1h-tetrazol-5-yl) butyloxy]-3, 4-dihydroquinolin-2(1H)-one) has been used worldwide to treat the symptoms of intermittent claudication caused by peripheral arterial disease. The InterSociety Consensus for the Management of Peripheral Arterial Disease (TASC II) has suggested cilostazol as the first-line drug for the relief of claudication symptoms because it improves treadmill exercise performance and quality of life.4 Cilostazol is a phosphodiesterase III inhibitor that suppresses degradation of cyclic adenosine monophosphate (cAMP), resulting in an increased concentration of cAMP in platelets and blood vessels, which leads to inhibition of platelet aggregation and vasodilation, respectively.5,6 In addition, cilostazol inhibits leukocyte adhesion to the endothelium7 and appears to tighten the endothelial barrier in situ by partly inhibiting cAMP-degrading enzymes in the endothelium.8 A study reported that cilostazol suppresses neointimal formation by suppressing proliferation of vascular smooth muscle cells.9 Recent studies have shown that cilostazol alleviates I/R injury in the brain, spinal cord, and small bowel.10e12 Previously, we investigated the mechanism of liver I/R injury.13e15 Based on our findings, in the present study, we hypothesized that cilostazol is beneficial for the treatment of liver I/R injury. We also examined the effect of cilostazol on the survival rate in rats with liver I/R injury.
Liver I/R injury was induced in the rats according to a previously reported procedure.16 Briefly, a midline incision was made after shaving the abdomen, and the abdominal cavity was then exposed with the aid of retractors. Next, the left hepatoduodenal ligament containing the hepatic artery, portal vein, and bile duct of the left lateral and median liver lobes was clamped for 60 or 90 min with a microvascular clamp to induce partial (70%) warm ischemia. The 90-min ischemia model was used to determine survival and the 60-min ischemia model was for all other experiments; 60 min of partial ischemia is nonlethal.17 Thereafter, the clip was removed to initiate hepatic reperfusion, and the abdominal cavity was closed. All the procedures were performed under general anesthesia induced with isoflurane. The rats were subjected to warm ischemia for 60 min followed by 3 h of reperfusion. The animals were then euthanized by total blood collection from the aorta by exsanguination. Liver tissues and blood samples were taken for analysis.
Materials and methods Animals Male Wistar rats aged 8 wk (weighing 250-300 g) were purchased from Japan SLC, Inc (Hamamatsu, Shizuoka, Japan) and housed in a temperature- and humiditycontrolled room under a 12-h light/dark cycle. The animals were given free access to water and food. All experiments were performed in accordance with the guidelines for the use of experimental animals by the National Institutes of Health and were approved by the Laboratory Animal Care and Use Committee of Keio University School of Medicine.
Experimental protocol The rats were randomly assigned to either a control group (I/R þ saline, n ¼ 15) or cilostazol group (I/R þ cilostazol, n ¼ 15). Cilostazol (Otsuka Pharmaceutical, Tokushima, Japan) was dissolved in a 0.5% carboxymethylcellulose sodium salt (Wako Pure Chemical Industries Ltd, Osaka, Japan) solution at a concentration of 10 mg/mL. Each rat was administered cilostazol (20 mg/kg/d) or saline (0.5 mL/ body/d) through a 3-Fr feeding tube (NIPRO, Osaka, Japan) into the stomach. These administrations were started 3 d before the surgery. On the day of the surgery, cilostazol or saline was administered 30 min before the induction of ischemia.18 Cilostazol or saline was administered 4 times before the surgery was performed (Fig. 1).
Laboratory measurements Blood samples were obtained from the aorta at the end of the experimental protocol and centrifuged at 3000 rpm for 10 min to obtain serum, which was immediately stored at 80 C. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were determined using ultraviolet spectrophotometric and enzymatic assays. We also measured serum interleukin (IL)-1b, IL-6, and tumor necrosis factor-a (TNF-a) levels using an R&D Rat Magnetic Luminex Screening Assay Kit (R&D Systems Inc, Minneapolis, MN).
Histologic examination Liver tissues for histologic examination were sampled from the left and median liver lobes at the end of the experimental protocol. The specimens were fixed in a 10% buffered formaldehyde solution, embedded in paraffin, and stained with hematoxylin and eosin. The tissue samples were sectioned into 5-mm-thick pieces for analysis. Histologic examination was performed based on randomly selected tissue sections (n ¼ 5 from each group) using the scoring system proposed by
209
fujii et al effect of cilostazol in liver i/r injury
saline
saline
saline
I/R injury
saline
0
1
2
Analysis of survival
3
Time after I/R injury (days) 0 CZ
CZ
0
1
2
2
6
7
6
7
CZ I/R injury
CZ
1
Analysis of survival
3
Time after I/R injury (days) 0
1
2
Fig. 1 e Experimental protocol. Rats were administered cilostazol (20 mg/kg/d) or saline (0.5 mL/body/d) via a feeding tube into the stomach once per day. These administrations were started 3 d before surgery. On the day surgery was performed, cilostazol or saline was administered 30 min before the induction of ischemia. The survival rate after inducing hepatic ischemia/reperfusion (I/R) was assessed every 24 h until 7 d after surgery.
Suzuki et al.19; specifically, the degree of sinusoidal congestion (none, minimal, mild, moderate, or severe), liver cell vacuolization (none, minimal, mild, moderate, or severe), and necrosis (none, single cell necrosis, < 30%, 30%-60%, or > 60%) in the tissues was determined. The histologic changes were scored from 0 to 4, and the total score, ranging from 0 to 12, was determined for each sample. All the sample slides were evaluated by the same pathologist, who was blinded to the corresponding control samples.
Survival analysis Eighteen Wistar rats (control, n ¼ 10; cilostazol, n ¼ 8) were used for a 7-d study on survival rate. Liver I/R injury was induced in the rats by subjecting them to warm ischemia for 90 min. After hepatic reperfusion, the rats recovered in individual cages and were fed a standard chow diet. Survival rate after the liver I/R injury was assessed every 24 h until 7 d after the operation (Fig. 1). The same cilostazol dosage (20 mg/kg/d) was administered in all the experiments.
Statistical analysis All results are expressed as means standard error of the mean. We performed between-group comparisons using either the ManneWhitney U test or Student t-test. A P value < 0.05 was considered statistically significant. We used the Kaplan-Meier analysis and log-rank testing to assess the
survival rate. All the statistical analyses were performed using SPSS 22 software (SPSS Inc, Chicago, IL).
Results Cilostazol preserves liver function To determine whether cilostazol alleviates liver I/R injury, cilostazol was administered to the rats before inducing warm I/R. As illustrated in Figure 2, serum AST (cilostazol versus control, 2427 1375 IU/L versus 4080 1870 IU/L; P ¼ 0.016) and ALT (cilostazol versus control, 2149 1299 versus 3513 1513 IU/L; P ¼ 0.023) levels at 3 h after hepatic reperfusion were significantly (P < 0.05) lower in the cilostazol group than in the control group. Therefore, treatment with cilostazol significantly alleviated liver I/R injury as indicated by the lowered levels of liver enzymes in the serum.
Cilostazol suppresses the expression of inflammatory cytokines in liver I/R injury Serum concentrations of IL-1b (cilostazol versus control, 31.7 26.2 pg/mL versus 49.0 19.0 pg/mL; P ¼ 0.013) and IL-6 (cilostazol versus control, 477 519 pg/mL versus 792 342 pg/ mL; P ¼ 0.020) were significantly lower in the cilostazol group than in the control group. However, no statistically significant differences were observed between serum TNF-a levels in the 2 groups (cilostazol versus control, 45.2 23.4 pg/mL versus 43.0 26.1 pg/mL; P ¼ 0.870; Fig. 3).
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Fig. 2 e Serum AST and ALT levels. Serum concentrations of AST and ALT were determined 3 h after hepatic reperfusion. The data are presented as means ± standard error of the mean (n [ 15 in each group).
Cilostazol improves the pathologic findings in liver I/R injury Histologic changes observed in the liver tissues are shown in Figure 4. In the control group, necrosis and sinusoidal congestions were evident from the periportal area to the midzonal area. In addition, vacuolization was apparent in the hepatocytes at the periphery of the necrotic area. In the cilostazol group, mild-to-moderate vacuolization and mild sinusoidal congestion were observed; however, no necrotic hepatocytes were observed (Fig. 4). The Suzuki scores for assessing liver damage were 3.4 1.1 and 6.0 2.1 for the cilostazol and saline control groups, respectively. The data indicate that liver damage was significantly (P < 0.05) alleviated by cilostazol. In particular, the Suzuki score for cytoplasmic vacuolization in the cilostazol group was significantly lower than that of the control group (cilostazol versus control, 1.4 0.5 versus 2.2 0.4; P ¼ 0.035). However, differences between Suzuki scores for sinusoidal congestion and necrosis in the 2 treatment groups were not statistically significant (Table).
Cilostazol increases the survival rate after liver I/R injury Treatment with cilostazol significantly improved the survival rate of rats after induction of liver I/R injury. The 7-d survival
rates were 63% and 100% in the saline control and cilostazol groups, respectively (P ¼ 0.038; Fig. 5).
Discussion Cilostazol is a quinolone compound that has antiplatelet effects. It acts directly on endothelial cells to inhibit the expression of adhesion molecules and inhibits neutrophil adhesion to the endothelium.20 After administration, cilostazol elevates the concentration of cAMP in endothelial cells and hepatocytes, which leads to the protection of endothelial cells and the maintenance of hepatic microcirculation and hepatocellular integrity.21e23 In rodent models, some studies have shown that I/R injury is alleviated through the cell protective effect of cilostazol in the kidney, heart, small intestine, limbs, muscles, spinal cord, cerebrum, and retina.12,24e28 It has been recently reported that intraperitoneally administered cilostazol alleviates hepatic I/R injury in mice.29 Although cilostazol was administered orally and intraperitoneally to rodents during these studies; clinically, it is only administered orally. In the present study, we analyzed the effects of oral cilostazol on survival rate and alleviation of hepatic injury in a rat model of partial warm hepatic I/R injury.
Fig. 3 e Serum levels of IL-1b, IL-6, and TNF-a. Serum concentrations of IL-1b, IL-6, and TNF-a were determined 3 h after hepatic reperfusion. The data are presented as means ± standard error of the mean (n [ 15 in each group).
fujii et al effect of cilostazol in liver i/r injury
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Fig. 4 e Histologic examination of the liver after I/R injury. Histologic analysis was performed on formalin-fixed liver sections (panels A and C are from the control group, whereas panels B and D are from the cilostazol group). The liver sections were obtained from the rats after they were subjected to partial hepatic ischemia for 60 min followed by 3 h of reperfusion. Representative images are shown. C, central vein and P, portal vein. Scale bars: 500 mm (A and B) and 100 mm (C and D).(Color version of figure is available online.)
Serum AST and ALT levels are the most objective indices of hepatocyte injury and are reported to markedly reflect the degree of hepatic I/R injury.30 Results of the present study show significant decreases in serum AST and ALT levels 3 h after reperfusion in the cilostazol group, suggesting that cilostazol alleviates hepatocyte injury. IL-1b and IL-6 are proinflammatory cytokines produced in Kupffer cells and hepatocytes after hepatic I/R injury.31 IL-1b is known to be closely associated with T cells/macrophages/neutrophils.32 In the present study, we observed significantly lower levels of serum IL-1b and IL-6 in the cilostazol group than in the saline group. These data suggest that alleviation of hepatic I/R injury
Table e Analysis of Suzuki scores. Histological changes
Control
Cilostazol
P value
Sinusoidal congestion
2.6 0.89
1.8 0.45
0.111
Cytoplasmic vacuolization
2.2 0.45
1.4 0.55
0.035
Necrosis
1.2 1.3
0.2 0.45
0.143
Total score
6.0 2.1
3.4 1.14
0.042
Estimation of the Suzuki score, which is representative of hepatocellular damage, was carried out 3 h after hepatic reperfusion. (n ¼ 5 in each group). Values are means standard error.
by cilostazol is mediated by the suppression of IL-1b and IL-6 expression levels. TNF-a expressed in Kupffer cells mediates hepatic I/R injury by inducing apoptosis and deterioration of the microcirculation through enhanced expression of cell adhesion molecules, which are induced by the generation of reactive oxygen species.31 However, in the present study, no significant differences were observed between serum TNF-a levels in the cilostazol and control groups. A study by Kim et al. reported that TNF-a levels peak after 6 h.33 Therefore, TNF-a levels at 6 h after the reperfusion in this model would be a next interesting. Hepatocellular vacuolization is a characteristic of cold hepatic I/R injury in rats. It has been reported that cilostazol suppresses the activation of endothelial cells and alleviates hepatocyte injury to reduce hepatocellular vacuolization.34 In contrast, in rat models of warm hepatic I/R injury, degeneration of nonparenchymal cells with vacuolization has been observed. Hepatic warm ischemia is poorly tolerated and rapidly leads to the death of sinusoidal endothelial cells and hepatocytes.23 The results of our study show that cilostazol improves these pathologic findings. Particularly, cytoplasmic vacuolization was markedly improved. This suggests that cilostazol alleviates hepatic I/R injury at the hepatocellular level. Other phosphodiesterase III inhibitors, including milrinone, olprinone, and amrinone are primarily used for managing cardiovascular diseases; however, they have been
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Fig. 5 e Analysis of survival rate after liver I/R injury. Survival rate was determined up to 7 d after 90 min of partial hepatic artery, portal vein, and bile duct clamping followed by reperfusion in rats (control group, n [ 8; cilostazol group, n [ 10).
shown to alleviate hepatic I/R injury in the liver and kidney.35e37Preconditioning is a technique conducted before hepatic ischemia to relieve I/R injury, whereas postconditioning is a protective procedure performed after I/R.38 Amrinone and milrinone have been noted to exert preconditioning and postconditioning effects in rats.39 Therefore, cilostazol treatment may result in a postconditioning effect in addition to the preconditioning effect observed in the present study. The data we obtained indicate that cilostazol improves survival rate after hepatic I/R injury. Other agents that have been observed to improve survival rate after hepatic I/R injury in rodent models include lithium and amrinone.37,40 Mortality may not reflect drug effects; rather, it may reflect events that are not ascertainable. Ischemia for 60 min is nonlethal17; therefore, we applied ischemia for 90 min to determine survival. In our preliminary experiments, survival rates were 100% 7 d after ischemia for 60 min. In the present study, the dosage of cilostazol administered to the rats was 20 mg/kg/d, which falls within the range of cilostazol dosages (10-40 mg/kg/d) administered to rats in previous studies.41,42 A previous study reported that oral administration of cilostazol at a dose of 10 mg/kg in rats is equivalent to a 100-mg/kg single dose in humans.43 Because the clinical dosage of cilostazol in humans is 200 mg/d, we administered 20 mg/kg/d to the rats. In the present study, the duration of induced hepatic partial ischemia was 60 min; however, previous studies have indicated that the extent of hepatic dysfunction is mild when the duration of ischemia is < 60 min.33,44 Pathologic findings were less remarkable for reperfusion lasting 3 h or less after ischemia; vacuolization, in
particular, remained relatively unchanged. However, when the reperfusion duration is > 3 h, necrosis and apoptosis become marked.44 We performed hepatic reperfusion over a period of 3 h in the present study and observed a significant improvement in vacuolization in the cilostazol group. In our preliminary experiments, we fixed the ischemic time to 1 h, and blood was collected 3, 6, 12, and 24 h after reperfusion. Hepatocyte damage, as shown by deviations in liver enzymes, peaked at 3 h. Consequently, we set the time after reperfusion at 3 h. According to Ritschel’s theory, drug concentrations reach a steady state when they are continuously administered 5 times longer than the half-life and the dose interval is less than 3 times the half-life. The half-life of cilostazol is 10-13 h; therefore, the time required for cilostazol to reach a steady state was estimated to be 50-65 h. We administered cilostazol for 3 d before reperfusion (72 h), and the experiment was conducted after the drug concentration reached a steady state. Although several agents have been reported to be effective in animal models of hepatic I/R injury, none have been clinically used in humans.31 Cilostazol is used clinically for the management of peripheral artery disease and for the prevention of cerebral infarction. Furthermore, the safety of cilostazol after oral administration has been established. Therefore, cilostazol may be a useful prophylactic against hepatic I/R injury. The primary limitation of the present study is the use of a rat model of partial (70%) hepatic ischemia induced by blocking blood flow to the median and left liver lobes to avoid enteric congestion. Therefore, the blood supply from the portal vein to nonischemic areas of the liver was higher than the blood supply to ischemic areas after reperfusion. This may have affected the extent of hepatic injury and rate of liver regeneration.45 In conclusion, the results of our study showed that oral administration of cilostazol before hepatic ischemia alleviates hepatic I/R injury in rats by protecting against hepatocyte injury and improving liver function, which can increase survival. Although further studies are required to understand the detailed mechanism by which cilostazol alleviates hepatic I/R injury, cilostazol may be a potential therapeutic agent for preventing hepatic I/R injury.
Acknowledgment The authors are grateful to Drs Yuko Matsuda and Kazumasa Fukuda for their invaluable technical assistance. Authors’ contributions: H.O. and T.F. were responsible for the conception and design of the project as well as collection of data. T.F., H.O., and M.S. analyzed the data. Y.M. and M.S. assessed the pathological findings. T.F., H.O., K.M., and N.F. were responsible for writing and revising the manuscript. T.F., H.O., K.M., N.F., H.Y., T.H., Y.A., M.K., M.S., O.I., M.T., Y.M., M.S., and Y.K. gave final approval of the manuscript.
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article.
fujii et al effect of cilostazol in liver i/r injury
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